Divergent Hemogen Genes of Teleosts and Mammals Share Conserved Roles in Erythropoiesis: Analysis Using Transgenic and Mutant Zebrafish Michael J
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© 2018. Published by The Company of Biologists Ltd | Biology Open (2018) 7, bio035576. doi:10.1242/bio.035576 RESEARCH ARTICLE Divergent Hemogen genes of teleosts and mammals share conserved roles in erythropoiesis: analysis using transgenic and mutant zebrafish Michael J. Peters, Sandra K. Parker, Jeffrey Grim*, Corey A. H. Allard‡, Jonah Levin§ and H. William Detrich, III¶ ABSTRACT (Krüger et al., 2002, 2005). Here we analyze the developmental Hemogen is a vertebrate transcription factor that performs important roles of teleost Hemogen using the zebrafish model system and its functions in erythropoiesis and testicular development and may powerful suite of reverse-genetic technologies. contribute to neoplasia. Here we identify zebrafish Hemogen and Teleost Hemogen was discovered using a subtractive show that it is considerably smaller (∼22 kDa) than its human ortholog hybridization screen designed to isolate novel erythropoietic (∼55 kDa), a striking difference that is explained by an underlying genes from fish belonging to the largely Antarctic suborder modular structure. We demonstrate that Hemogens are largely Notothenioidei (Detrich and Yergeau, 2004; Yergeau et al., composed of 21-25 amino acid repeats, some of which may function 2005). Sixteen species belonging to the icefish family as transactivation domains (TADs). Hemogen expression in embryonic (Channichthyidae) are unique among vertebrates because they are and adult zebrafish is detected in hematopoietic, renal, neural and white-blooded; they fail to execute the erythroid genetic program or gonadal tissues. Using Tol2- and CRISPR/Cas9-generated transgenic produce hemoglobin (Cocca et al., 1995; Near et al., 2006; Zhao zebrafish, we show that Hemogen expression is controlled by two et al., 1998). Forty-five candidate erythropoietic cDNAs were Gata1-dependent regulatory sequences that act alone and together to recovered using representational difference analysis (Hubank and control spatial and temporal expression during development. Partial Schatz, 1999) applied to kidney marrow transcriptomes of two depletion of Hemogen in embryos by morpholino knockdown reduces notothenioid species, one red-blooded and the other white-blooded the number of erythrocytes in circulation. CRISPR/Cas9-generated (Detrich and Yergeau, 2004; Yergeau et al., 2005). One of the zebrafish lines containing either a frameshift mutation or an in-frame unknown genes, clone Rda130, was similar to mammalian deletion in a putative, C-terminal TAD display anemia and embryonic Hemogen and was expressed only by the red-blooded notothenioid. tail defects. This work expands our understanding of Hemogen and Although Hemogen is clearly involved in hematopoiesis, its provides mutant zebrafish lines for future study of the mechanism of mechanism remains incompletely understood. In human cell lines, this important transcription factor. Hemogen activates erythroid gene transcription in part by recruiting the histone acetyltransferase P300 to acetylate Gata1 (Zheng et al., KEY WORDS: Anemia, CRISPR/Cas9, Gene editing, Hematopoiesis, 2014). Like Gata1, Hemogen protects erythroid cells from apoptosis Transcription by upregulating anti-apoptotic factors (e.g. Nf-κB, Bcl-xL) that are critical for terminal differentiation (Li et al., 2004; Rhodes et al., INTRODUCTION 2005; Zhang et al., 2012). Hemogen (Hemgn) is a vertebrate transcription factor that is The regulation of Hemogen expression is of interest because it is expressed in mammalian hematopoietic progenitors (Lu et al., 2001; overexpressed frequently in patients with a variety of cancers and Yang et al., 2001) and has been implicated in erythroid leukemias (An et al., 2005; Forbes et al., 2017; Li et al., 2004). This differentiation and survival (Li et al., 2004). Originally identified putative oncogene, which is located in a human chromosomal in mice and subsequently described in humans as EDAG region (9q22) of leukemia-associated breakpoints, has been linked (Erythrocyte Differentiation Associated Gene), Hemogen has also to proliferation and survival of leukemic cells and to induction of been implicated in testis development in mammals and chickens tumor formation in mice (Chen et al., 2016; Lu et al., 2002). Thus, (Nakata et al., 2013; Yang et al., 2003), and in osteogenesis in rats somatic mutations in Hemogen or its regulators may contribute to neoplasia. The zebrafish is a well-established model organism for studying Department of Marine and Environmental Sciences, Northeastern University, Nahant, MA 01908, USA. hematopoiesis in vertebrates because it produces the same blood *Present address: Department of Biology, The University of Tampa, Tampa, FL lineages as mammals (de Jong and Zon, 2005; Paffett-Lugassy and ‡ 33606, USA. Present address: Department of Biochemistry and Cell Biology, Zon, 2005). In zebrafish, erythropoiesis occurs in sequential waves at Geisel School of Medicine at Dartmouth College, Hanover, NH 03755, USA. §Present address: Department of Biochemistry, McGill University, Montreal, unique anatomical locations in embryos and adults that correspond to Quebec H3G1Y6, Canada. analogous sites in mammals (Galloway and Zon, 2003). Many of the ¶Author for correspondence ([email protected]) molecular players that orchestrate the erythroid program appear to be conserved between zebrafish and mammals, but relatively few have M.J.P., 0000-0003-4932-528X; J.G., 0000-0002-3344-0657; C.A.H.A., 0000- been functionally characterized in zebrafish. Nevertheless, mutant 0002-3554-2059; H.W.D., 0000-0002-0783-4505 zebrafish models accurately phenocopy human blood diseases caused This is an Open Access article distributed under the terms of the Creative Commons Attribution by mutations in major erythroid factors, such as Gata1 (Lyons et al., License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 2002) and Erythroid beta-spectrin (Liao et al., 2000). The purpose of this study is to characterize the regulation of Received 7 May 2018; Accepted 16 July 2018 Hemogen expression and the function of the Hemogen protein in Biology Open 1 RESEARCH ARTICLE Biology Open (2018) 7, bio035576. doi:10.1242/bio.035576 Fig. 1. Zebrafish Si:dkey-25o16.2 and human Hemogen are orthologous and encode related proteins that differ in size. (A) Structure of the zebrafish Hemogen-like gene, Si:dkey-25o16.2. Two conserved noncoding elements (C1 and C2, black boxes) were identified in a 2 kb segment proximal to the start codon (see Results, Figs 4-6). Coding exons, white boxes; noncoding exons, gray boxes. Numbers indicate length in bp. (B) Synteny of loci for zebrafish Si:dkey-25o16.2 on chromosome 1 and Hemogen on human chromosome 9 (region q22). Transcriptional orientations indicated by arrows. (C) Alternative splicing of zebrafish Hemogen-like transcripts showing sequenced regions. Introns are shown as chevrons. Transcripts 1 and 2 differ by retention of 12 bp of intron (red). (D) Modular structures of zebrafish and human Hemogen proteins each encoded by four exons (numbered boxes). Locations of truncating mutations found in some human cancers (Forbes et al., 2017) are indicated by asterisks. Predicted regions and motifs: green, coiled coil; blue, nuclear localization signal; red, four residues introduced by alternative splicing; yellow, tandem peptide repeats; brown, acidic repeat with transactivation domain (TAD) motif; gray, no prediction. (E) Three-dimensional ab initio models of Hemogens. The ribbon diagram of the zebrafish protein, color-coded as in panel D, is superimposed on the gray, space-filling model for the human protein (See Materials and Methods). zebrafish. We identify the zebrafish Hemogen ortholog, which predicted gene Si:dkey-25o16.2 on chromosome 1 of the zebrafish despite being only 40% as large as the human protein, contains genome (Howe et al., 2013). When we compared the synteny of the similarly arranged functional motifs. Hemogen is expressed in putative teleost and mammalian orthologs, represented in Fig. 1B blood, testis, ovaries, kidneys and the central nervous system in by zebrafish Si:dkey-25o16.2 (chromosome Dr1) and human zebrafish. Two tissue-specific, alternative Hemogen promoters Hemogen (chromosome Hs9), we found that the flanking genes are associated with conserved noncoding elements (CNEs) and and their transcriptional orientations were conserved, which have distinct regulatory functions in primitive and definitive strongly supported Si:dkey-25o16.2 as the zebrafish Hemogen hematopoiesis and other processes. By analysis of morphant and ortholog. mutant zebrafish, we show that Hemogen is required for normal erythropoiesis and that this role depends in part on a cluster of acidic Structure of the zebrafish Hemogen gene residues within a putative, C-terminal transactivation domain The basic structure of the Hemogen gene of teleosts and mammals (TAD). was also found to be highly conserved; four coding exons were separated by three introns (Fig. 1A), and two introns were found in RESULTS the 5′-UTR. Two transcription start sites were predicted to occur Teleosts contain a single Hemogen-like gene that is syntenic within 2 kb upstream of the Hemogen start codon in zebrafish with human Hemogen (Fig. 1A) and these appear to correspond to the hematopoietic- and Chromosomal synteny is an important criterion when assigning testis-specific Hemogen promoters (noncoding exons 1H and 1T, gene relationships across divergent